Dysfunction of cobalamin transport and metabolism, if left untreated, will result in neurological disorder and megablastic anemia. The purpose of this program is to characterize the transport and metabolism of cobalamin at the molecular level. Cobalamin-binding proteins (e.g., serum transcobalamin II) which mediate membrane transport have been purified to homogeneity by affinity chromatography. Cellular receptors for transcobalamin II have been identified on murine leukemia L1210 cells with the use of immobilized forms of the protein. Thus, latex particles (0.35 micrometer) containing covalently attached transcobalamin II bind to receptors localized in cellular microvilli. The particles are internalized by the cells via clathrin-coated pits and vesicles. Intracellular transport (and hence metabolism) of cobalamins may be directed by the serum transport proteins after the internalization process. The availability of pure cobalamin-binding proteins and a rat-liver perfusion system is providing information on this problem. The conversion of methylcobalamin and cyanocobalamin to adenosylcobalamin is being studied in the cobalamin auxotroph Lactobacillus leichmannii. The initial step in cobalamin metabolism is the removal of the upper axial ligand (CN- or -CH3). Using an extremely sensitive assay system (solid phase aminoundecylcobalamin (57 Co), the enzyme (s) catalyzing this first step in cobalamin metabolism has been detected in crude extracts; purification is in progress. The enzymes responsible for the next two steps in cobalamin metabolism, namely reduction and adenosylation, are also being purified and characterized. A similar pathway is being investigated in mammalian cells (L1210 and rat liver) and organelles.